The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common species infecting this site. Most data regarding expression of uropathogenic E. coli (UPEC) virulence genes has come from in vitro studies or the murine model of urinary tract infection (UTI). It is not clear how gene expression in the murine model compares to gene expression during a clinical UTI in humans. Lack of such knowledge may prevent research that focuses on the most highly expressed virulence mechanisms and even lead to work not relevant to human infection. Our long term research goal is to understand how UPEC colonize the human urinary tract, avoid the immune response, and damage the host. The objective during this funding period is to determine the levels of expression of the three classes of UPEC genes during clinical UTI in women that most affect virulence: adhesin, iron acquisition system, and toxin genes. Our central hypothesis is that the virulence of a UPEC strain is the sum of its capacity for adherence, iron acquisition, and toxin production. The rationale for the proposed work is that once we identify the most highly expressed virulence genes during UTI in humans, we can focus efforts on intervention directed toward these highly expressed targets. We will achieve our objective by completing the following specific aims: 1) Determine the adhesins expressed during UTI in humans;2) determine the iron acquisition systems required for cystitis and those required for acute pyelonephritis;and 3) Determine the toxins expressed during human UTI that predict virulence. The expected outcomes of conducting these three aims will be a precise assessment of virulence gene expression by UPEC strains during recurrent and uncomplicated UTI in women. Given a specific virulence gene profile, we will be able to predict the potential for virulence of that particular strain. The positive impact of these studies will be substantial. We will precisely define the nature of a UPEC strain as they differ from commensal strains. We will understand which virulence determinants including adhesins, iron acquisition system proteins, and toxins are expressed during clinical UTI in women and to what degree they are expressed. At the conclusion of our project we will have definitively quantified global gene expression in UPEC strains during acute UTI in women. These findings will be critical to the field because they will identify virulence determinants expressed by UPEC during UTI in women that will clarify the mechanism of pathogenesis, classify therapeutic targets, and highlight antigens with potential for vaccine development. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because urinary tract infection (UTI) remains a significant source of morbidity. Most recently there were 8.2 million physician visits, over 1.7 million emergency room visits, and 366,000 hospitalizations of both men and women in the United States for UTI, at an annual cost of 3.4 billion dollars. These frequencies place UTIs first among kidney and urologic diseases in terms of total cost. This is relevant to NIH's mission because precisely defining the mechanism by which E. coli infects the urinary tract will lead to therapies that may extend healthy life by reducing the burden of these infections.